TWEAK functions with TNF and IL-17 on keratinocytes and is a potential target for psoriasis therapy

Abstract

TNF and IL-17 are two cytokines that drive dysregulated keratinocyte activity, and their targeting is highly efficacious in patients with psoriasis, but whether these molecules act with other inflammatory factors is not clear. Here, we show that mice having a keratinocyte-specific deletion of Fn14 (Tnfrsf12a), the receptor for the TNF superfamily cytokine TWEAK (Tnfsf12), displayed reduced imiquimod-induced skin inflammation, including diminished epidermal hyperplasia and less expression of psoriasis signature genes. This corresponded with Fn14 being expressed in keratinocytes in human psoriasis lesions and TWEAK being found in several subsets of skin cells. Transcriptomic studies in human keratinocytes revealed that TWEAK strongly overlaps with IL-17A and TNF in up-regulating the expression of CXC chemokines, along with cytokines such as IL-23 and inflammation-associated proteins like S100A8/9 and SERPINB1/B9, all previously found to be highly expressed in the lesional skin of patients with psoriasis. TWEAK displayed strong synergism with TNF or IL-17A in up-regulating messenger RNA for many psoriasis-associated genes in human keratinocytes, including IL23A, IL36G, and multiple chemokines, implying that TWEAK acts with TNF and IL-17 to enhance feedback inflammatory activity. Correspondingly, therapeutic treatment of mice with anti-TWEAK was equally as effective as antibodies to IL-17A or TNF in reducing clinical and immunological features of psoriasis-like skin inflammation and combination targeting of TWEAK with either cytokine had no greater inhibitory effect, reinforcing the conclusion that all three cytokines function together. Thus, blocking TWEAK could be comparable to targeting TNF or IL-17 and might be considered as an alternate therapeutic treatment for psoriasis.

Fig. 1.. TWEAK and Fn14 are expressed in cells from psoriasis lesions and Fn14 keratinocyte-specific conditional knockout mice are protected from imiquimod-induced skin inflammation.

(A-B) UMAPs showing Seurat-normalized expression levels (left) and percentage of cells that are positive per cluster (right) for (A) TNFSF12 and (B) TNFRSF12A in CD45+ (left) and CD45− (right) cells in human psoriasis lesions (see Fig. S1 for cluster annotations). (C-F) Fn14 flox and Fn14 cKO mice were treated with imiquimod on the back for 7 days and compared to naive untreated mice. (C) Representative H&E stained skin sections. Arrows highlight epidermis thickness. Average epidermal thickness (μM), calculated from 18-20 random measurements, from two experiments with 5 mice/group. Each point represents one mouse. (D) IF staining for Ki-67 (pink) in the epidermis and quantification of Ki-67+ cells/mm. (E) IF staining for KRT14+ cells (pink) in the epidermis and mean fluorescence intensity measurement. (F) mRNA expression of indicated genes in skin biopsies. Data fold change over naïve group. Individual mice or means ± SEM from 3-8 mice/group, combined from 2 experiments. *P <0.05, **P<0.01, ***P<0.001.

Fig. 2.. TWEAK induces inflammatory activity in human keratinocytes overlapping with IL-17 and TNF.

(A-C) RNA-seq of human keratinocytes from triplicates stimulated with human TWEAK, IL-17A, or TNF. (A) Venn diagram comparing the numbers of upregulated unique and overlapping transcripts common to the human psoriasis biopsy transcriptome. (B, C) Heatmap and boxplot analysis showing the transcript patterns of the 129 psoriatic genes upregulated by TWEAK. (D-E) RNA-seq of skin from mice subcutaneously injected with mouse TWEAK. (D) Venn diagram showing the number of transcripts upregulated in the skin common to the human psoriasis biopsy transcriptome. (E) Venn diagram showing the number of transcripts upregulated in the skin that were common to the human psoriasis biopsy transcriptome compared to the number upregulated by TWEAK in human keratinocytes.

Fig. 3.. TWEAK and TNF synergize to upregulate multiple psoriasis-relevant gene transcripts in human keratinocytes.

RNA-seq of human keratinocytes from triplicates stimulated with TWEAK or TNF alone or in combination. (A) Venn diagram showing the number of transcripts synergistically upregulated by TWEAK and TNF common to the human psoriasis biopsy transcriptome. (B, C) Heatmap and boxplot analysis showing the expression pattern of the 95 synergistically upregulated psoriatic genes. Several key psoriasis genes highlighted. (D) Experimental fold change for select psoriasis genes synergistically upregulated by TWEAK and TNF.

Fig. 4.. TWEAK and IL-17A synergize to upregulate multiple psoriasis-relevant gene transcripts in human keratinocytes.

RNA-seq of human keratinocytes from triplicates stimulated with TWEAK or IL-17A alone or in combination. (A) Venn diagram showing the number of transcripts synergistically upregulated by TWEAK and IL-17A common to the human psoriasis biopsy transcriptome. (B, C) Heatmap and boxplot analysis showing the expression pattern of the 13 synergistically upregulated psoriatic genes. (D) Experimental fold change for select psoriasis genes synergistically upregulated by TWEAK and IL-17A.

Fig. 5.. Therapeutic blockade of TWEAK reduces clinical and immunological features of imiquimod-driven skin inflammation similar to blockade of IL-17A.

WT mice were treated with imiquimod on the dorsal skin for 11 constitutive days. Mice received anti-TWEAK or anti-IL-17A alone or together, or control IgG, on days 6 and 9, with analysis on day 12. (A) Representative H&E stained skin tissue sections, and quantitation of epidermal thickness in individual mice. (B) IF staining and enumeration of Ki-67+ cells/mm of epidermis in individual mice. (C) Immune cell infiltrates in skin biopsies showing the number of leukocytes, neutrophils, macrophages, and γδ T cells. (D) mRNA expression of indicated genes in skin biopsies. GAPDH was considered as an endogenous control. Data mean fold change ± SEM over naïve group. (A-C) Data from 6-10 mice/group; (D) Data from 4-6 mice/group, combined from 2 experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6.. Therapeutic blockade of TWEAK is comparable to blockade of TNF in reducing features of imiquimod-induced skin inflammation.

WT mice were treated with imiquimod on the dorsal skin for 11constitutive days. Mice received anti-TWEAK or anti-TNF alone or together, or control IgG, on days 6 and 9, with analysis on day 12. (A) Representative H&E stained skin tissue sections, and quantitation of epidermal thickness in individual mice. (B) IF staining and enumeration of Ki-67+ cells/mm of epidermis in individual mice. (C) Immune cell infiltrates in skin biopsies showing the number of leukocytes, neutrophils, macrophages, and γδ T cells. (D) mRNA expression of indicated genes in skin biopsies. GAPDH used as an endogenous control. Data mean fold change ± SEM over naïve group. (A-C) Data from 6-10 mice/group; (D) Data from 4-6 mice/group, combined from 2 experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.